Burdens of Ascaris spp. and Cryptosporidium spp. parasites in farm pigs in Ghana

Abstract Background Worldwide, intestinal parasites significantly affect the health and production of pigs. Objective This study assessed the prevalence of Ascaris and Cryptosporidium infection in pigs in the Ejisu‐Juaben Municipality of Ghana. Method Faecal samples from two hundred (200) pigs on four different farms (labelled A, B, C, D) were processed using the Kinyoun modified Ziehl‐Neelsen method for Cryptosporidium and the Formol‐ether sedimentation method for Ascaris and microscopically examined to identify parasites to the genus level. Results The prevalence of Ascaris and Cryptosporidium in the pigs was 76% and 77%, respectively. The weaners had the highest Ascaris prevalence (96.15%) with the piglets recording the least (59.25%). On the other hand, the piglets had the highest prevalence (88.89%) for Cryptosporidium with the boars, sows and weaners recording 75.86%, 75.42% and 73.08% respectively. The prevalence of Ascaris was high in farm D (78.57%) while Cryptosporidium was highest in farm C (86.11%). Generally, there was a significant difference (p = 0.044) in the mean distribution of Cryptosporidium in the pigs. Conclusion The high burden of Ascaris and Cryptosporidium infections in the pigs suggest the need to adopt and implement effective control measures.

Gastrointestinal parasites greatly influence the productivity of pigs and other livestock industries by causing substantial economic loss (Boes et al., 2000). Generally, the parasites are transmitted via an oral-faecal route through infected food and water. The humid and warm conditions of the tropics as well as the deficient treatment of local pigs against parasitic diseases (Mashatise et al., 2005) invariably cause them to carry heavy burdens of gastrointestinal parasites. It is evident through various research findings of the widespread especially in Sub-Saharan Africa, gastrointestinal parasitic infections in pigs (Nwafor et al., 2019;Omoruyi & Agbinone, 2020;Youssao et al., 2006).
In the case of Ascaris, human infections are known to be caused by Ascaris lumbricoides (Ali et al., 2020) while infections in pigs are caused by Ascaris suum (Zheng et al., 2020). However, the interaction between humans and pigs has resulted in cross-species transmission (Anderson, 1995;Monteiro et al., 2019;Sadaow et al., 2018) with interbreeding between Ascaris lumbricoides and Ascaris suum (Criscione et al., 2007;Peng & Criscione, 2012). More studies are required to establish the zoonotic transmission of Ascaris as it is unclear if pigs are significant reservoirs of human infection (Da Silva Alves et al., 2016;Leles et al., 2012).
The Ejisu-Juaben Municipality is among the largest pig-breeding sites in the Ashanti Region. It is a place where pigs are sold in large commercial numbers for people in Ejisu-Juaben Municipality and even extends to reach people outside the Municipality. However, there is little to no information on the prevalence of Ascaris and Cryptosporidium among pigs in the Region. Thus, this study aimed at determining the prevalence of these intestinal parasites and further suggests the need to create and implement control measures.

Sample collection
The cross-sectional study was conducted in four intensive pig farms labelled as A, B, C and D in the Ejisu-Juaben Municipality. The pig farms were conveniently selected based on farm size (between 50 and 80 pigs per farm) and accessibility for faecal sampling. An intensive management farming system was seen among all the farms visited.
Fresh faecal samples were collected aseptically from the pigs into separate sterile zip-lock bags, labelled, preserved in 10% formalin and then carried to the laboratory for morphological examination of the parasites. Pigs under the age of 3 months were grouped as piglets, those within 3-6 months old were grouped as weaners and pigs 7 months and above were grouped as adult boars and adult sows. A total of two hundred (200)

Macroscopic and microscopic examination
Stool samples were examined by direct observation for mucus, blood, consistency (formed, soft or loose) and any adult parasites.
Examination of stool samples for Ascaris was done using the Formolether concentration technique (Cheesbrough, 2006). To 2 g of a stool sample, 10 ml of formalin was added; mixture stirred using an applicator stick until a slightly cloudy suspension was attained and 4 ml of ethyl acetate added to the suspension and mixed properly for 1 min. The faecal suspension was sieved into a centrifuge tube and centrifuged for 1 min at 3000 rpm. The debris plug was loosed with an applicator stick and the supernatant decanted. One drop of the sample was placed on a clean microscope slide without any gross fibres and particles. Immediately, 1 drop of Lugol's iodine was then added and covered with a coverslip. The specimen was then examined with the low power objective lens (10×) beginning at one corner of the smear and systematically examined successive adjacent swaths with the high-power objective lens (40×) for the eggs of Ascaris.
The Kinyoun modified Ziehl-Neelsen method was used in the preparation of samples for identification of Cryptosporidium (El-Moamly & El-Sweify, 2012). Briefly, a few drops (one to two) of the specimen were then smeared on the slide and allowed to air dry. It was then fixed with absolute methanol for 1 min and dried at room temperature. Set up was flooded with Kinyoun's carbol fuchsin for 5 min. The slide was rinsed briefly (3-5 s) with 50% ethanol and thoroughly with water thereafter. The smear was decolourized with 1% sulphuric acid for 2 min or until no more colour ran from the slide. The slide was rinsed with water, drained, counterstained with methylene blue for 1 min and rinsed with water. The slide was allowed to stand for airdrying. The prepared slide was finally mounted under the oil immersion objective for the morphological examination of Cryptosporidium oocyst.

Statistical analysis
Results were organized using Microsoft Excel spreadsheet 2010 and analysed for significant differences between the mean distribution of egg/cyst per gram of stool (Ascaris and Cryptosporidium respectively) in pigs across the farms using ANOVA with SPSS IBMS 2.0v. The prevalence was calculated for all data sets as the number of infected

RESULTS
Out of the 200 pig samples examined, an overall prevalence of 77% of Cryptosporidium infection and 76% of Ascaris infection in pigs were recorded. (Table 1). A significant difference was observed in the mean distribution of Cryptosporidium in the pigs (p = 0.044). It was observed that boars and piglets were mostly infected with Cryptosporidium whereas sows and weaners were mostly infected with Ascaris ( Figure 1).

Prevalence of Cryptosporidium spp. and Ascaris spp. across various pigs in farms
The prevalence of Cryptosporidium spp. and Ascaris spp. were recorded categorically in boars, sows, weaners and piglets (Figures 2 and 3).
Prevalence within the various farms showed no significant difference (p > 0.05).

Mean distribution of cyst/eggs of Cryptosporidium spp. and Ascaris spp. in pigs across the farms
The mean distribution of Cryptosporidium spp. in the various pig groups saw the highest infection in the boars of farm D, sows of farm C, weaners of farm A and piglets of farm A ( Table 2).
The mean distribution of Ascaris spp. in the various pig groups saw the highest infection in the boars of farm D, sows of farm A, weaners of farm B and piglets of farm C (Table 3).

DISCUSSION
Gastrointestinal parasites cause significant problems in pig farming by affecting pig health, increasing morbidity in younger animals and extreme cases of death (Kagira et al., 2012;Pinilla et al., 2020). Studies suggest that Ascaris and Cryptosporidium infections in pigs could be of public health importance (Cavallero et al., 2013;Zhang et al., 2013); thus there is a need to establish the current prevalence in pigs and the potential risk to farmers.
In Ghana, C. hominis and C. parvum have been identified in children within the Ashanti Region (Eibach et al., 2015). Furthermore, Cryp- that C parvum is a zoonotic pathogen (Adegbola et al., 1994;Yu & Seo, 2004) hence livestock could play the role of reservoirs in the spread of infections.
In this study, even though there was no significant difference between the Cryptosporidium prevalence recorded from the various farms (p > 0.05), it was observed that piglets were mostly infected with Cryptosporidium. It has been established that Cryptosporidium oocyst can survive for longer periods in the faecal matter as compared to bacterial pathogens (Hutchison et al., 2005). Thus, a lack of efficient hygienic practices on the farm could enhance the spread of this parasite. In addition, piglets being in close confinement with the sows could facilitate the easy transmission of infection from the sow (Fablet, 2009). The use of water from a well and borehole on the farms could also influence the distribution of Cryptosporidium (Karanis et al., 2007).
Although Ascaris lumbricoides infects humans (Ali et al., 2020) and Ascaris suum infects pigs (Zheng et al., 2020), there is evidence of crossspecies transmission between humans and pigs within the same location (Anderson, 1995;Monteiro et al., 2019;Sadaow et al., 2018). Furthermore, Ascaris lumbricoides and Ascaris suum can interbreed, posing a serious threat to public health (Criscione et al., 2007;Peng & Criscione, 2012  It is important to note that the high prevalence of Ascaris infections in pigs is due to the large number of eggs produced and their ability to survive over a longer period (Hagel & Giusti, 2010). Poor environmental hygiene is said to also maintain or increase the intensity of Ascaris infection (Stothard et al., 2008). Additionally, Cryptosporidium oocysts have a protective wall that facilitates their survival in water and other environments (Thompson et al., 2008).
This same protective coat makes the oocyst resistant to chlorination in water treatment (Bichai et al., 2008) and medical control of the oocysts in the pigs becomes difficult. Thus, to improve the health of pigs and increase production, it is necessary to adopt effective control measures against intestinal parasite infections and prevent or reduce transmission to farmers as well as exposure to the environment.

CONCLUSION
The study revealed a high prevalence of Cryptosporidium and Ascaris among pigs in the various farms. These parasites affect animal health and could potentially be transmitted to humans; thus, there is a need of establishing control measures to reduce the burden of infections. Furthermore, it is suggested that molecular studies be carried out in the pig farms to determine the specific species of intestinal parasites causing infections.